Methods for producing modified glycoproteins

ABSTRACT

Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins in humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans, including unicellular and multicellular fungi are modified to produce N-glycans such as Man 5 GlcNAc 2  or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/892,591, filedJun. 27, 2001, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/214,358, filed on Jun. 28, 2000, U.S. ProvisionalApplication Ser. No. 60/215,638, filed Jun. 30, 2000, and U.S.Provisional Application Ser. No. 60/279,997, filed on Mar. 30, 2001.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions by whichfungi or other eukaryotic microorganisms can be genetically modified toproduce glycosylated proteins (glycoproteins) having patterns ofglycosylation similar to glycoproteins produced by animal cells,especially human cells, which are useful as human or animal therapeuticagents.

BACKGROUND OF THE INVENTION

Glycosylation Pathways

De novo synthesized proteins may undergo further processing in cells,known as post-translational modification. In particular, sugar residuesmay be added enzymatically, a process known as glycosylation. Theresulting proteins bearing covalently linked oligosaccharide side chainsare known as glycosylated proteins or glycoproteins. Bacteria typicallydo not glycosylate proteins; in cases where glycosylation does occur itusually occurs at nonspecific sites in the protein (Moens andVanderleyden, Arch. Microbiol. 1997 168(3):169-175).

Eukaryotes commonly attach a specific oligosaccharide to the side chainof a protein asparagine residue, particularly an asparagine which occursin the sequence Asn-Xaa-Ser/Thr/Cys (where Xaa represents any aminoacid). Following attachment of the saccharide moiety, known as anN-glycan, further modifications may occur in vivo. Typically thesemodifications occur via an ordered sequence of enzymatic reactions,known as a cascade. Different organisms provide different glycosylationenzymes (glycosyltransferases and glycosidases) and different glycosylsubstrates, so that the final composition of a sugar side chain may varymarkedly depending upon the host.

For example, microorganisms such as filamentous fungi and yeast (lowereukaryotes) typically add additional mannose and/or mannosylphosphatesugars. The resulting glycan is known as a “high-mannose” type or amannan. By contrast, in animal cells, the nascent oligosaccharide sidechain may be trimmed to remove several mannose residues and elongatedwith additional sugar residues that typically do not occur in theN-glycans of lower eukaryotes. See R. K. Bretthauer, et al.Biotechnology and Applied Biochemistry, 1999, 30, 193-200; W. Martinet,et al. Biotechnology Letters, 1998, 20, 1171-1177; S. Weikert, et al.Nature Biotechnology, 1999, 17, 1116-1121; M. Malissard, et al.Biochemical and Biophysical Research Communications, 2000, 267, 169-173;Jarvis, et al. 1998 Engineering N-glycosylation pathways in thebaculovirus-insect cell system, Current Opinion in Biotechnology,9:528-533; and M. Takeuchi, 1997 Trends in Glycoscience andGlycotechnology, 1997, 9, S29-S35.

The N-glycans that are produced in humans and animals are commonlyreferred to as complex N-glycans. A complex N-glycan means a structurewith typically two to six outer branches with a sialyllactosaminesequence linked to an inner core structure Man₃GlcNAc₂. A complexN-glycan has at least one branch, and preferably at least two, ofalternating GlcNAc and galactose (Gal) residues that terminate inoligosaccharides such as, for example: NeuNAc-; NeuAcα2-6GalNAcα1-;NeuAcα2-3Galβ1-3GalNAcα1-; NeuAcα2-3/6Galβ1-4GlcNAcα1-;GlcNAcα1-4Galβ1-(mucins only); Fucα1-2Galβ1-(blood group H). Sulfateesters can occur on galactose, GalNAc, and GlcNAc residues, andphosphate esters can occur on mannose residues. NeuAc (Neu: neuraminicacid; Ac:acetyl) can be O-acetylated or replaced by NeuG1(N-glycolylneuraminic acid). Complex N-glycans may also have intrachainsubstitutions of bisecting GlcNAc and core fucose (Fuc).

Human glycosylation begins with a sequential set of reactions in theendoplasmatic reticulum (ER) leading to a core oligosaccharidestructure, which is transferred onto de novo synthesized proteins at theasparagine residue in the sequence Asn-Xaa-Ser/Thr (see FIG. 1A).Further processing by glucosidases and mannosidases occurs in the ERbefore the nascent glycoprotein is transferred to the early Golgiapparatus, where additional mannose residues are removed byGolgi-specific 1,2-mannosidases. Processing continues as the proteinproceeds through the Golgi. In the medial Golgi a number of modifyingenzymes including N-acetylglucosamine transferases (GnT I, GnT II, GnTIII, GnT IV GnT V GnT VI), mannosidase II, fucosyltransferases add andremove specific sugar residues (see FIG. 1B). Finally in the transGolgi, the N-glycans are acted on by galactosyl tranferases andsialyltransferases (ST) and the finished glycoprotein is released fromthe Golgi apparatus. The protein N-glycans of animal glycoproteins havebi-, tri-, or tetra-antennary structures, and may typically includegalactose, fucose, and N-acetylglucosamine. Commonly the terminalresidues of the N-glycans consist of sialic acid. A typical structure ofa human N-glycan is shown in FIG. 1B.

Sugar Nucleotide Precursors

The N-glycans of animal glycoproteins typically include galactose,fucose, and terminal sialic acid. These sugars are not generally foundon glycoproteins produced in yeast and filamentous fungi. In humans, thefull range of nucleotide sugar precursors (e.g. UDP-N-acetylglucosamine,UDP-N-acetylgalactosamine, CMP-N-acetylneuraminic acid, UDP-galactose,GDP-fucose etc.) are generally synthesized in the cytosol andtransported into the Golgi, where they are attached to the coreoligosaccharide by glycosyltransferases. (Sommers and Hirschberg, 1981J. Cell Biol. 91(2): A406-A406; Sommers and Hirschberg 1982 J. Biol.Chem. 257(18): 811-817; Perez and Hirschberg 1987 Methods in Enzymology138: 709-715.

Glycosyl transfer reactions typically yield a side product which is anucleoside diphosphate or monophosphate. While monophosphates can bedirectly exported in exchange for nucleoside triphosphate sugars by anantiport mechanism, diphosphonucleosides (e.g. GDP) have to be cleavedby phosphatases (e.g. GDPase) to yield nucleoside monophosphates andinorganic phosphate prior to being exported. This reaction is importantfor efficient glycosylation; for example, GDPase from S. cerevisiae hasbeen found to be necessary for mannosylation. However the GDPase has 90%reduced activity toward UDP (Beminsone et al., 1994 J. Biol. Chem.269(l):207-211α). Lower eukaryotes typically lack UDP-specificdiphosphatase activity in the Golgi since they do not utilize UDP-sugarprecursors for Golgi-based glycoprotein synthesis. Schizosaccharomycespombe, a yeast found to add galactose residues to cell wallpolysaccharides (from UDP-galactose) has been found to have specificUDPase activity, indicating the requirement for such an enzyme(Beminsone et al., 1994). UDP is known to be a potent inhibitor ofglycosyltransferases and the removal of this glycosylation side productis important in order to prevent glycosyltransferase inhibition in thelumen of the Golgi (Khatara et al., 1974). See Beminsone, P., et al.1995. J. Biol. Chem. 270(24): 14564-14567; Beaudet, L., et al. 1998 AbcTransporters: Biochemical, Cellular, and Molecular Aspects. 292:397-413.

Compartmentalization of Glycosylation Enzymes

Glycosyltransferases and mannosidases line the inner (luminal) surfaceof the ER and Golgi apparatus and thereby provide a catalytic surfacethat allows for the sequential processing of glycoproteins as theyproceed through the ER and Golgi network. The multiple compartments ofthe cis, medial, and trans Golgi and the trans Golgi Network (TGN),provide the different localities in which the ordered sequence ofglycosylation reactions can take place. As a glycoprotein proceeds fromsynthesis in the ER to full maturation in the late Golgi or TGN, it issequentially exposed to different glycosidases, mannosidases andglycosyltransferases such that a specific N-glycan structure may besynthesized. The enzymes typically include a catalytic domain, a stemregion, a membrane spanning region and an N-terminal cytoplasmic tail.The latter three structural components are responsible for directing aglycosylation enzyme to the appropriate locus.

Localization sequences from one organism may function in otherorganisms. For example the membrane spanning region ofα-2,6-sialyltransferase (α-2,6-ST) from rats, an enzyme known tolocalize in the rat trans Golgi, was shown to also localize a reportergene (invertase) in the yeast Golgi (Schwientek, et al., 1995). However,the very same membrane spanning region as part of a full-length ofα-2,6-sialyltransferase was retained in the ER and not furthertransported to the Golgi of yeast (Krezdom et al., 1994). A full lengthGalT from humans was not even synthesized in yeast, despite demonstrablyhigh transcription levels. On the other hand the transmembrane region ofthe same human GaIT fused to an invertase reporter was able to directlocalization to the yeast Golgi, albeit it at low production levels.Schwientek and co-workers have shown that fusing 28 amino acids of ayeast mannosyltransferase (Mnt1), a region containing an N-terminalcytoplasmic tail, a transmembrane region and eight amino acids of thestem region, to the catalytic domain of human GalT are sufficient forGolgi localization of an active GalT (Schwientek et al. 1995 J. Biol.Chem. 270(10):5483-5489). Other galactosyltransferases appear to rely oninteractions with enzymes resident in particular organelles since afterremoval of their transmembrane region they are still able to localizeproperly.

Improper localization of a glycosylation enzyme may prevent properfunctioning of the enzyme in the pathway. For example Aspergillusnidulans, which has numerous α-1,2-mannosidases (Eades and Hintz, 2000Gene 255(1):25-34), does not add GlcNAc to Man₅GlcNAc₂ when transformedwith the rabbit GnT I gene, despite a high overall level of GnT Iactivity (Kalsner et al., 1995). GnT I, although actively expressed, maybe incorrectly localized such that the enzyme is not in contact withboth of its substrates: the nascent N-glycan of the glycoprotein andUDP-GlcNAc. Alternatively, the host organism may not provide an adequatelevel of UDP-GlcNAc in the Golgi.

Glycoproteins Used Therapeutically

A significant fraction of proteins isolated from humans or other animalsare glycosylated. Among proteins used therapeutically, about 70% areglycosylated. If a therapeutic protein is produced in a microorganismhost such as yeast, however, and is glycosylated utilizing theendogenous pathway, its therapeutic efficiency is typically greatlyreduced. Such glycoproteins are typically immunogenic in humans and showa reduced half-life in vivo after administration (Takeuchi, 1997).

Specific receptors in humans and animals can recognize terminal mannoseresidues and promote the rapid clearance of the protein from thebloodstream. Additional adverse effects may include changes in proteinfolding, solubility, susceptibility to proteases, trafficking,transport, compartmentalization, secretion, recognition by otherproteins or factors, antigenicity, or allergenicity. Accordingly, it hasbeen necessary to produce therapeutic glycoproteins in animal hostsystems, so that the pattern of glycosylation is identical or at leastsimilar to that in humans or in the intended recipient species. In mostcases a mammalian host system, such as mammalian cell culture, is used.

Systems for Producing Therapeutic Glycoproteins

In order to produce therapeutic proteins that have appropriateglycoforms and have satisfactory therapeutic effects, animal orplant-based expression systems have been used. The available systemsinclude:

-   -   1. Chinese hamster ovary cells (CHO), mouse fibroblast cells and        mouse myeloma cells (Arzneimittelforschung. 1998        August;48(8):870-880);    -   2. transgenic animals such as goats, sheep, mice and others        (Dente Prog. Clin. Biol. 1989 Res. 300:85-98, Ruther et al.,        1988 Cell 53(6):847-856; Ware, J., et al. 1993 Thrombosis and        Haemostasis 69(6): 1194-1194; Cole, E. S., et al. 1994 J. Cell.        Biochem. 265-265);    -   3. plants (Arabidopsis thaliana, tobacco etc.) (Staub, et al.        2000 Nature Biotechnology 18(3): 333-338) (McGarvey, P. B., et        al. 1995 Bio-Technology 13(13): 1484-1487; Bardor, M., et al.        1999 Trends in Plant Science 4(9): 376-380);    -   4. insect cells (Spodoptera frugiperda Sf9, Sf21, Trichoplusia        ni, etc. in combination with recombinant baculoviruses such as        Autographa californica multiple nuclear polyhedrosis virus which        infects lepidopteran cells) (Altmans et al., 1999 Glycoconj. J.        16(2):109-123).

Recombinant human proteins expressed in the above-mentioned host systemsmay still include non-human glycoforms (Raju et al., 2000 AnnalsBiochem. 283(2):123-132). In particular, fraction of the N-glycans maylack terminal sialic acid, typically found in human glycoproteins.Substantial efforts have been directed to developing processes to obtainglycoproteins that are as close as possible in structure to the humanforms, or have other therapeutic advantages. Glycoproteins havingspecific glycoforms may be especially useful, for example in thetargeting of therapeutic proteins. For example, the addition of one ormore sialic acid residues to a glycan side chain may increase thelifetime of a therapeutic glycoprotein in vivo after admininstration.Accordingly, the mammalian host cells may be genetically engineered toincrease the extent of terminal sialic acid in glycoproteins expressedin the cells. Alternatively sialic acid may be conjugated to the proteinof interest in vitro prior to administration using a sialic acidtransferase and an appropriate substrate. In addition, changes in growthmedium composition or the expression of enzymes involved in humanglycosylation have been employed to produce glycoproteins more closelyresembling the human forms (S. Weikert, et al., Nature Biotechnology,1999, 17, 1116-1121; Werner, Noe, et al 1998 Arzneimittelforschung48(8):870-880; Weikert, Papac et al., 1999; Andersen and Goochee 1994Cur. Opin. Biotechnol.5: 546-549; Yang and Butler 2000 Biotechnol.Bioengin. 68(4): 370-380). Alternatively cultured human cells may beused.

However, all of the existing systems have significant drawbacks. Onlycertain therapeutic proteins are suitable for expression in animal orplant systems (e.g. those lacking in any cytotoxic effect or othereffect adverse to growth). Animal and plant cell culture systems areusually very slow, frequently requiring over a week of growth undercarefully controlled conditions to produce any useful quantity of theprotein of interest. Protein yields nonetheless compare unfavorably withthose from microbial fermentation processes. In addition cell culturesystems typically require complex and expensive nutrients and cofactors,such as bovine fetal serum. Furthermore growth may be limited byprogrammed cell death (apoptosis).

Moreover, animal cells (particularly mammalian cells) are highlysusceptible to viral infection or contamination. In some cases the virusor other infectious agent may compromise the growth of the culture,while in other cases the agent may be a human pathogen rendering thetherapeutic protein product unfit for its intended use. Furthermore manycell culture processes require the use of complex,temperature-sensitive, animal-derived growth media components, which maycarry pathogens such as bovine spongiform encephalopathy (BSE) prions.Such pathogens are difficult to detect and/or difficult to remove orsterilize without compromising the growth medium. In any case, use ofanimal cells to produce therapeutic proteins necessitates costly qualitycontrols to assure product safety.

Transgenic animals may also be used for manufacturing high-volumetherapeutic proteins such as human serum albumin, tissue plasminogenactivator, monoclonal antibodies, hemoglobin, collagen, fibrinogen andothers. While transgenic goats and other transgenic animals (mice,sheep, cows, etc.) can be genetically engineered to produce therapeuticproteins at high concentrations in the milk, the process is costly sinceevery batch has to undergo rigorous quality control. Animals may host avariety of animal or human pathogens, including bacteria, viruses,fungi, and prions. In the case of scrapies and bovine spongiformencephalopathy, testing can take about a year to rule out infection. Theproduction of therapeutic compounds is thus preferably carried out in awell-controlled sterile environment, e.g. under Good ManufacturingPractice (GMP) conditions. However, it is not generally feasible tomaintain animals in such environments. Moreover, whereas cells grown ina fermenter are derived from one well characterized Master Cell Bank(MCB), transgenic animal technology relies on different animals and thusis inherently non-uniform. Furthermore external factors such asdifferent food uptake, disease and lack of homogeneity within a herd,may effect glycosylation patterns of the final product. It is known inhumans, for example, that different dietary habits result in differingglycosylation patterns.

Transgenic plants have been developed as a potential source to obtainproteins of therapeutic value. However, high level expression ofproteins in plants suffers from gene silencing, a mechanism by which thegenes for highly expressed proteins are down-regulated in subsequentplant generations. In addition, plants add xylose and/or α-1,3-linkedfucose to protein N-glycans, resulting in glycoproteins that differ instructure from animals and are immunogenic in mammals (Altmann, Marz etal., 1995 Glycoconj. J. 12(2);150-155). Furthermore, it is generally notpractical to grow plants in a sterile or GMP environment, and therecovery of proteins from plant tissues is more costly than the recoveryfrom fermented microorganisms.

Glycoprotein Production Using Eukaryotic Microorganisms

The lack of a suitable expression system is thus a significant obstacleto the low-cost and safe production of recombinant human glycoproteins.Production of glycoproteins via the fermentation of microorganisms wouldoffer numerous advantages over the existing systems. For example,fermentation-based processes may offer (a) rapid production of highconcentrations of protein; (b) the ability to use sterile,well-controlled production conditions (e.g. GMP conditions); (c) theability to use simple, chemically defined growth media; (d) ease ofgenetic manipulation; (e) the absence of contaminating human or animalpathogens; (f) the ability to express a wide variety of proteins,including those poorly expressed in cell culture owing to toxicity etc.;(g) ease of protein recovery (e.g. via secretion into the medium). Inaddition, fermentation facilities are generally far less costly toconstruct than cell culture facilities.

As noted above, however, bacteria, including species such as Escherichiacoli commonly used to produce recombinant proteins, do not glycosylateproteins in a specific manner like eukaryotes. Various methylotrophicyeasts such as Pichia pastoris, Pichia methanolica, and Hansenulapolymorpha, are particularly useful as eukaryotic expression systems,since they are able to grow to high cell densities and/or secrete largequantities of recombinant protein. However, as noted above,glycoproteins expressed in these eukaryotic microorganisms differsubstantially in N-glycan structure from those in animals. This hasprevented the use of yeast or filamentous fungi as hosts for theproduction of many useful glycoproteins.

Several efforts have been made to modify the glycosylation pathways ofeukaryotic microorganisms to provide glycoproteins more suitable for useas mammalian therapeutic agents. For example, severalglycosyltransferases have been separately cloned and expressed in S.cerevisiae (GalT, GnT I), Aspergillus nidulans (GnT I) and other fungi(Yoshida et al., 1999, Kalsner et al., 1995 Glycoconj. J. 12(3):360-370,Schwientek et al., 1995). However, N-glycans with human characteristicswere not obtained.

Yeasts produce a variety of mannosyltransferases e.g.1,3-mannosyltransferases (e.g. MNN1 in S. cerevisiae) (Graham and Emr,1991 J. Cell. Biol. 114(2):207-218), 1,2-mannosyltransferases (e.g.KTR/KRE family from S. cerevisiae), 1,6-mannosyltransferases (OCH1 fromS. cerevisiae), mannosylphosphate transferases (MNN4 and MNN6 from S.cerevisiae) and additional enzymes that are involved in endogenousglycosylation reactions. Many of these genes have been deletedindividually, giving rise to viable organisms having alteredglycosylation profiles. Examples are shown in Table 1. TABLE 1 Examplesof yeast strains having altered mannosylation N-glycan (wild N-glycanStrain type) Mutation (mutant) Reference S. pombe Man_(>9)GlcNAc₂ OCH1Man₈GlcNAc₂ Yoko-o et al., 2001 FEBS Lett. 489(1): 75-80 S. cerevisiaeMan_(>9)GlcNAc₂ OCH1/MNN1 Man₈GlcNAc₂ Nakanishi-Shindo et al,. 1993 J.Biol. Chem. 268(35): 26338-26345 S. cerevisiae Man_(>9)GlcNAc₂OCH1/MNN1/MNN4 Man₈GlcNAc₂ Chiba et al., 1998 J. Biol. Chem. 273,26298-26304

In addition, Japanese Patent Application Public No. 8-336387 disclosesan OCH1 mutant strain of Pichia pastoris. The OCH1 gene encodes1,6-mannosyltransferase, which adds a mannose to the glycan structureMan₈GlcNAc₂ to yield Man₉GlcNAc₂. The Man₉GlcNAc₂ structure is then asubstrate for further mannosylation in vivo, leading to thehypermannosylated glycoproteins that are characteristic of yeasts andtypically may have at least 30-40 mannose residue per N-glycan. In theOCH1 mutant strain, proteins glycosylated with Man₈GlcNAc₂ areaccumulated and hypermannosylation does not occur. However, thestructure Man₈GlcNAc₂ is not a substrate for animal glycosylationenzymes, such as human UDP-GlcNAc transferase I, and accordingly themethod is not useful for producing proteins with human glycosylationpatterns.

Martinet et al. (Biotechnol. Lett. 1998, 20(12), 1171-1177) reported theexpression of α-1,2-mannosidase from Trichoderma reesei in P. pastoris.Some mannose trimming from the N-glycans of a model protein wasobserved. However, the model protein had no N-glycans with the structureMan₅GlcNAc₂, which would be necessary as an intermediate for thegeneration of complex N-glycans. Accordingly the method is not usefulfor producing proteins with human or animal glycosylation patterns.

Similarly, Chiba et al. 1998 expressed a-1,2-mannosidase fromAspergillus saitoi in the yeast Saccharomyces cerevisiae. A signalpeptide sequence (His-Asp-Glu-Leu) was engineered into the exogenousmannosidase to promote retention in the endoplasmic reticulum. Inaddition, the yeast host was a mutant lacking three enzyme activitiesassociated with hypermannosylation of proteins: 1,6-mannosyltransferase(OCH1); 1,3-mannosyltransferase (MNN1); and mannosylphosphatetransferase(MNN4). The N-glycans of the triple mutant host thus consisted of thestructure Man₈GlcNAc₂, rather than the high mannose forms found inwild-type S. cerevisiae. In the presence of the engineered mannosidase,the N-glycans of a model protein (carboxypeptidase Y) were trimmed togive a mixture consisting of 27 mole % Man₅GlcNAc₂, 22 mole %Man₆GlcNAc₂, 22 mole % Man₇GlcNAc₂, 29 mole % Man₈GlcNAc₂. Trimming ofthe endogenous cell wall glycoproteins was less efficient, only 10 mole% of the N-glycans having the desired Man₅GlcNAc₂ structure.

Since only the Man₅GlcNAc₂ glycans would be susceptible to furtherenzymatic conversion to human glycoforms, the method is not efficientfor the production of proteins having human glycosylation patterns. Inproteins having a single N-glycosylation site, at least 73 mole % wouldhave an incorrect structure. In proteins having two or threeN-glycosylation sites, respectively at least 93 or 98 mole % would havean incorrect structure. Such low efficiencies of coversion areunsatisfactory for the production of therapeutic agents, particularly asthe separation of proteins having different glycoforms is typicallycostly and difficult.

With the object of providing a more human-like glycoprotein derived froma fungal host, U.S. Pat. No. 5,834,251 to Maras and Contreras disclosesa method for producing a hybrid glycoprotein derived from Trichodermareesei. A hybrid N-glycan has only mannose residues on the Manα1-6 armof the core and one or two complex antennae on the Manα1-3 arm. Whilethis structure has utility, the method has the disadvantage thatnumerous enzymatic steps must be performed in vitro, which is costly andtime-consuming. Isolated enzymes are expensive to prepare and maintain,may need unusual and costly substrates (e.g. UDP-GlcNAc), and are proneto loss of activity and/or proteolysis under the conditions of use.

It is therefore an object of the present invention to provide a systemand methods for humanizing glycosylation of recombinant glycoproteinsexpressed in Pichia pastoris and other lower eukaryotes such asHansenula polymorpha, Pichia stiptis, Pichia methanolica, Pichia sp,Kluyveromyces sp, Candida albicans, Aspergillus nidulans, andTrichoderma reseei.

SUMMARY OF THE INVENTION

Cell lines having genetically modified glycosylation pathways that allowthem to carry out a sequence of enzymatic reactions, which mimic theprocessing of glycoproteins in humans, have been developed. Recombinantproteins expressed in these engineered hosts yield glycoproteins moresimilar, if not substantially identical, to their human counterparts.The lower eukaryotes, which ordinarily produce high-mannose containingN-glycans, including unicellular and multicellular fungi such as Pichiapastoris, Hansenula polymorpha, Pichia stiptis, Pichia methanolica,Pichia sp., Kluyveromyces sp., Candida albicans, Aspergillus nidulans,and Trichoderma reseei, are modified to produce N-glycans such asMan₅GlcNAc₂ or other structures along human glycosylation pathways. Thisis achieved using a combination of engineering and/or selection ofstrains which: do not express certain enzymes which create theundesirable complex structures characteristic of the fungalglycoproteins, which express exogenous enzymes selected either to haveoptimal activity under the conditions present in the fungi whereactivity is desired, or which are targeted to an organelle where optimalactivity is achieved, and combinations thereof wherein the geneticallyengineered eukaryote expresses multiple exogenous enzymes required toproduce “human-like” glycoproteins.

In a first embodiment, the microorganism is engineered to express anexogenous α-1,2-mannosidase enzyme having an optimal pH between 5.1 and8.0, preferably between 5.9 and 7.5. In an alternative preferredembodiment, the exogenous enzyme is targeted to the endoplasmicreticulum or Golgi apparatus of the host organism, where it trimsN-glycans such as Man₈GlcNAc₂ to yield Man₅GlcNAc₂. The latter structureis useful because it is identical to a structure formed in mammals,especially humans; it is a substrate for further glycosylation reactionsin vivo and/or in vitro that produce a finished N-glycan that is similaror identical to that formed in mammals, especially humans; and it is nota substrate for hypermannosylation reactions that occur in vivo in yeastand other microorganisms and that render a glycoprotein highlyimmunogenic in animals.

In a second embodiment, the glycosylation pathway of an eukaryoticmicroorganism is modified by (a) constructing a DNA library including atleast two genes encoding exogenous glycosylation enzymes; (b)transforming the microorganism with the library to produce a geneticallymixed population expressing at least two distinct exogenousglycosylation enzymes; (c) selecting from the population a microorganismhaving the desired glycosylation phenotype. In a preferred embodiment,the DNA library includes chimeric genes each encoding a proteinlocalization sequence and a catalytic activity related to glycosylation.Organisms modified using the method are useful for producingglycoproteins having a glycosylation pattern similar or identical tomammals, especially humans.

In a third embodiment, the glycosylation pathway is modified to expressa sugar nucleotide transporter enzyme. In a preferred embodiment, anucleotide diphosphatase enzyme is also expressed. The transporter anddiphosphatase improve the efficiency of engineered glycosylation steps,by providing the appropriate substrates for the glycosylation enzymes inthe appropriate compartments, reducing competitive product inhibition,and promoting the removal of nucleoside diphosphates.

DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic diagram of typical fungal N-glycosylationpathway.

FIG. 1B is a schematic diagram of a typical human N-glycosylationpathway.

DETAILED DESCRIPTION OF THE INVENTION

The methods and recombinant lower eukaryotic strains described hereinare used to make “humanized glycoproteins”. The recombinant lowereukaryotes are made by engineering lower eukaryotes which do not expressone or more enzymes involved in production of high mannose structures toexpress the enzymes required to produce human-like sugars. As usedherein, a lower eukaryote is a unicellular or filamentous fungus. Asused herein, a “humanized glycoprotein” refers to a protein havingattached thereto N-glycans including less than four mannose residues,and the synthetic intermediates (which are also useful and can bemanipulated further in vitro) having at least five mannose residues. Ina preferred embodiment, the glycoproteins produced in the recombinantlower eukaryotic strains contain at least 27 mole % of the Man5intermediate. This is achieved by cloning in a better mannosidase, i.e.,an enzyme selected to have optimal activity under the conditions presentin the organisms at the site where proteins are glycosylated, or bytargeting the enzyme to the organelle where activity is desired.

In a preferred embodiment, eukaryotic strains which do not express oneor more enzymes involved in the production of high mannose structuresare used. These strains can be engineered or one of the many suchmutants already described in yeasts, including ahypermannosylation-minus (OCH1) mutant in Pichia pastoris.

The strains can be engineered one enzyme at a time, or a library ofgenes encoding potentially useful enzymes can be created, and thosestrains having enzymes with optimal activities or producing the most“human-like” glycoproteins, selected.

Lower eukaryotes that are able to produce glycoproteins having theattached N-glycan Man₅GlcNAc₂ are particularly useful since (a) lackinga high degree of mannosylation (e.g. greater than 8 mannoses perN-glycan, or especially 30-40 mannoses), they show reducedimmunogenicity in humans; and (b) the N-glycan is a substrate forfurther glycosylation reactions to form an even more human-likeglycoform, e.g. by the action of GlcNAc transferase I to formGlcNAcMan₅GlcNAc₂. Man₅GlcNAc₂ must be formed in vivo in a high yield,at least transiently, since all subsequent glycosylation reactionsrequire Man₅GlcNAc₂ or a derivative thereof. Accordingly, a yield isobtained of greater than 27 mole %, more preferably a yield of 50-100mole %, glycoproteins in which a high proportion of N-glycans haveMan₅GlcNAc₂. It is then possible to perform further glycosylationreactions in vitro, using for example the method of U.S. Pat. No.5,834,251 to Maras and Contreras. In a preferred embodiment, at leastone further glycosylation reaction is performed in vivo. In a highlypreferred embodiment thereof, active forms of glycosylating enzymes areexpressed in the endoplasmic reticulum and/or Golgi apparatus.

Host Microorganisms

Yeast and filamentous fungi have both been successfully used for theproduction of recombinant proteins, both intracellular and secreted(Cereghino, J. L. and J. M. Cregg 2000 FEMS Microbiology Reviews 24(1):45-66; Harkki, A., et al. 1989 Bio-Technology 7(6): 596; Berka, R. M.,et al. 1992 Abstr. Papers Amer. Chem. Soc. 203: 121-BIOT; Svetina, M.,et al. 2000 J. Biotechnol. 76(2-3): 245-251.

Although glycosylation in yeast and fungi is very different than inhumans, some common elements are shared. The first step, the transfer ofthe core oligosaccharide structure to the nascent protein, is highlyconserved in all eukaryotes including yeast, fungi, plants and humans(compare FIGS. 1A and 1B). Subsequent processing of the coreoligosaccharide, however, differs significantly in yeast and involvesthe addition of several mannose sugars. This step is catalyzed bymannosyltransferases residing in the Golgi (e.g. OCH1, MNT1, MNN1,etc.), which sequentially add mannose sugars to the coreoligosaccharide. The resulting structure is undesirable for theproduction of humanoid proteins and it is thus desirable to reduce oreliminate mannosyl transferase activity. Mutants of S. cerevisiae,deficient in mannosyl transferase activity (e.g. och1 or mnn9 mutants)have shown to be non-lethal and display a reduced mannose content in theoligosacharide of yeast glycoproteins. Other oligosacharide processingenzymes, such as mannosylphophate transferase may also have to beeliminated depending on the host's particular endogenous glycosylationpattern. After reducing undesired endogenous glycosylation reactions theformation of complex N-glycans has to be engineered into the hostsystem. This requires the stable expression of several enzymes andsugar-nucleotide transporters. Moreover, one has to locate these enzymesin a fashion such that a sequential processing of the maturingglycosylation structure is ensured.

Target Glycoproteins

The methods described herein are useful for producing glycoproteins,especially glycoproteins used therapeutically in humans. Suchtherapeutic proteins are typically administered by injection, orally,pulmonary, or other means.

Examples of suitable target glycoproteins include, without limitation:erythropoietin, cytokines such as interferon-α, interferon-β,interferon-γ, interferon-ω, and granulocyte-CSF, coagulation factorssuch as factor VIII, factor IX, and human protein C, soluble IgEreceptor α-chain, IgG, IgM, urokinase, chymase, and urea trypsininhibitor, IGF-binding protein, epidermal growth factor, growthhormone-releasing factor, annexin V fusion protein, angiostatin,vascular endothelial growth factor-2, myeloid progenitor inhibitoryfactor-1, and osteoprotegerin.

Method for Producing Glycoproteins Comprising the N-glycan Man₅GlcNAc₂

The first step involves the selection or creation of a lower eukaryotethat is able to produce a specific precursor structure of Man₅GlcNAc₂,which is able to accept in vivo GlcNAc by the action of a GlcNActransferase I. This step requires the formation of a particular isomericstructure of Man₅GlcNAc₂. This structure has to be formed within thecell at a high yield (in excess of 30%) since all subsequentmanipulations are contingent on the presence of this precursor.Man₅GlcNAc₂ structures are necessary for complex N-glycan formation,however, their presence is by no means sufficient, since Man₅GlcNAc₂ mayoccur in different isomeric forms, which may or may not serve as asubstrate for GlcNAc transferase I. Most glycosylation reactions are notcomplete and thus a particular protein generally contains a range ofdifferent carbohydrate structures (i.e. glycoforms) on its surface. Themere presence of trace amounts (less than 5%) of a particular structurelike Man₅GlcNAc₂ is of little practical relevance. It is the formationof a particular, GlcNAc transferase I accepting intermediate (StructureI) in high yield (above 30%), which is required. The formation of thisintermediate is necessary and subsequently allows for the in vivosynthesis of complex N-glycans.

One can select such lower eukaryotes from nature or alternativelygenetically engineer existing fungi or other lower eukaryotes to providethe structure in vivo. No lower eukaryote has been shown to provide suchstructures in vivo in excess of 1.8% of the total N-glycans (Maras etal., 1997), so a genetically engineered organism is preferred. Methodssuch as those described in U.S. Pat. No. 5,595,900, may be used toidentify the absence or presence of particular glycosyltransferases,mannosidases and sugar nucleotide transporters in a target organism ofinterest.

Inactivation of Fungal Glycosylation Enzymes such as1,6-mannosyltransferase

The method described herein may be used to engineer the glycosylationpattern of a wide range of lower eukaryotes (e.g. Hansenula polymorpha,Pichia stiptis, Pichia methanolica, Pichia sp, Kluyveromyces sp, Candidaalbicans, Aspergillus nidulans, Trichoderma reseei etc.). Pichiapastoris is used to exemplify the required manipulation steps. Similarto other lower eukaryotes, P. pastoris processes Man₉GlcNAc₂ structuresin the ER with a 1,2-α-mannosidase to yield Man₈GlcNAc₂. Through theaction of several mannosyltransferases, this structure is then convertedto hypermannosylated structures (Man_(>9)GlcNAc2), also known asmannans. In addition, it has been found that P. pastoris is able to addnon-terminal phosphate groups, through the action of mannosylphosphatetransferases to the carbohydrate structure. This is contrary to thereactions found in mammalian cells, which involve the removal of mannosesugars as opposed to their addition. It is of particular importance toeliminate the ability of the fungus to hypermannosylate the existingMan₈GlcNAc₂ structure. This can be achieved by either selecting for afungus that does not hypermannosylate, or by genetically engineeringsuch a fungus.

Genes that are involved in this process have been identified in Pichiapastoris and by creating mutations in these genes one is able to reducethe production of “undesirable” glycoforms. Such genes can be identifiedby homology to existing mannosyltransferases (e.g. OCH1, MNN4, MNN6,MNN1), found in other lower eukaryotes such as C. albicans, Pichiaangusta or S. cerevisiae or by mutagenizing the host strain andselecting for a phenotype with reduced mannosylation. Based onhomologies amongst known mannosyltransferases and mannosylphosphatetransferases, one may either design PCR primers, examples of which areshown in Table 2, or use genes or gene fragments encoding such enzymesas probes to identify homologues in DNA libraries of the targetorganism. Alternatively, one may be able to complement particularphenotypes in related organisms. For example, in order to obtain thegene or genes encoding 1,6-mannosyltransferase activity in P. pastoris,one would carry out the following steps. OCH 1 mutants of S. cerevisiaeare temperature sensitive and are slow growers at elevated temperatures.One can thus identify functional homologues of OCH1 in P. pastoris bycomplementing an OCH1 mutant of S. cerevisiae with a P. pastoris DNA orcDNA library. Such mutants of S. cerevisiae may be found e.g., see theSaccharomyces genome link at the Stanford University website and arecommercially available. Mutants that display a normal growth phenotypeat elevated temperature, after having been transformed with a P.pastoris DNA library, are likely to carry an OCH1 homologue of P.pastoris. Such a library can be created by partially digestingchromosomal DNA of P. pastoris with a suitable restriction enzyme andafter inactivating the restriction enzyme ligating the digested DNA intoa suitable vector, which has been digested with a compatible restrictionenzyme. Suitable vectors are pRS314, a low copy (CEN6/ARS4) plasmidbased on pBluescript containing the Trp1 marker (Sikorski, R. S., andHieter, P.,1989, Genetics 122, pg 19-27) or pFL44S, a high copy (2μ)plasmid based on a modified pUC19 containing the URA3 marker (Bonneaud,N., et al., 1991, Yeast 7, pg. 609-615). Such vectors are commonly usedby academic researchers or similar vectors are available from a numberof different vendors such as Invitrogen (Carlsbad, Calif.), Pharmacia(Piscataway, N.J.), New England Biolabs (Beverly, Mass.). Examples arepYES/GS, 2μ origin of replication based yeast expression plasmid fromInvitrogen, or Yep24 cloning vehicle from New England Biolabs. Afterligation of the chromosomal DNA and the vector one may transform the DNAlibrary into strain of S. cerevisiae with a specific mutation and selectfor the correction of the corresponding phenotype. After sub-cloning andsequencing the DNA fragment that is able to restore the wild-typephenotype, one may use this fragment to eliminate the activity of thegene product encoded by OCH1 in P. pastoris.

Alternatively, if the entire genomic sequence of a particular fungus ofinterest is known, one may identify such genes simply by searchingpublicly available DNA databases, which are available from severalsources such as NCBI, Swissprot etc. For example by searching a givengenomic sequence or data base with a known 1,6mannosyltransferase gene(OCH1) from S. cerevisiae, one can able to identify genes of highhomology in such a genome, which a high degree of certainty encodes agene that has 1,6mannosyltransferase activity. Homologues to severalknown mannosyltransferases from S. cerevisiae in P. pastoris have beenidentified using either one of these approaches. These genes havesimilar functions to genes involved in the mannosylation of proteins inS. cerevisiae and thus their deletion may be used to manipulate theglycosylation pattern in P. pastoris or any other fungus with similarglycosylation pathways.

The creation of gene knock-outs, once a given target gene sequence hasbeen determined, is a well-established technique in the yeast and fungalmolecular biology community, and can be carried out by anyone ofordinary skill in the art (R. Rothsteins, (1991) Methods in Enzymology,vol. 194, p. 281). In fact, the choice of a host organism may beinfluenced by the availability of good transformation and genedisruption techniques for such a host. If several mannosyltransferaseshave to be knocked out, the method developed by Alani and Klecknerallows for the repeated use of the URA3 markers to sequentiallyeliminate all undesirable endogenous mannosyltransferase activity. Thistechnique has been refined by others but basically involves the use oftwo repeated DNA sequences, flanking a counter selectable marker. Forexample: URA3 may be used as a marker to ensure the selection of atransformants that have integrated a construct. By flanking the URA3marker with direct repeats one may first select for transformants thathave integrated the construct and have thus disrupted the target gene.After isolation of the transformants, and their characterization, onemay counter select in a second round for those that are resistant to5′FOA. Colonies that able to survive on plates containing 5′FOA havelost the URA3 marker again through a crossover event involving therepeats mentioned earlier. This approach thus allows for the repeateduse of the same marker and facilitates the disruption of multiple geneswithout requiring additional markers.

Eliminating specific mannosyltransferases, such as1,6mannosyltransferase (OCH1), mannosylphosphate transferases (MNN4,MNN6, or genes complementing lbd mutants) in P. pastoris, allows for thecreation of engineered strains of this organism which synthesizeprimarily Man₈GlcNAc₂ and thus can be used to further modify theglycosylation pattern to more closely resemble more complex humanglycoform structures. A preferred embodiment of this method utilizesknown DNA sequences, encoding known biochemical glycosylation activitiesto eliminate similar or identical biochemical functions in P. pastoris,such that the glycosylation structure of the resulting geneticallyaltered P. pastoris strain is modified. TABLE 2 Target Gene(s) in P. PCRprimer A PCR primer B pastoris Homologues ATGGCGAAGGCAGA TTAGTCCTTCCAAC1,6- OCH1 S. TGGCAGT TTCCTTC mannosyl- cerevisiae, (SEQ ID NO: 1) (SEQID NO: 2) trans- Pichia ferase albicans TAYTGGMGNGTNGA GCRTCNCCCCANCK1,2 KTR/KRE RCYNGAYATHAA YTCRTA mannosyl- family, (SEQ ID NO: 3) (SEQ IDNO: 4) trans- S. ferases cerevisiaeLegend: M = A or C, R = A or G, W = A or T, S = C or G, Y = C or T, K =G or T, V = A or C or G, H = A or C or T, D = A or G or T, B = C or G orT, N = G or A or T or C.

Incorporation of a Mannosidase into the Genetically Engineered Host

The process described herein enables one to obtain such a structure inhigh yield for the purpose of modifying it to yield complex N-glycans. Asuccessful scheme to obtain suitable Man₅GlcNAc₂ structures must involvetwo parallel approaches: (1) reducing endogenous mannosyltransferaseactivity and (2) removing 1,2-α-mannose by mannosidases to yield highlevels of suitable Man₅GlcNAc₂ structures. What distinguishes thismethod from the prior art is that it deals directly with those twoissues. As the work of Chiba and coworkers demonstrates, one can reduceMan₈GlcNAc₂ structures to a Man₅GlcNAc₂ isomer in S. cerevisiae, byengineering the presence of a fungal mannosidase from A. saitoi into theER. The shortcomings of their approach are twofold: (1) insufficientamounts of Man₅GlcNAc₂ are formed in the extra-cellular glycoproteinfraction (10%) and (2) it is not clear that the in vivo formedMan₅GlcNAc₂ structure in fact is able to accept GlcNAc by action ofGlcNAc transferase I. If several glycosylation sites are present in adesired protein the probability (P) of obtaining such a protein in acorrect form follows the relationship P=(F)^(n), where n equals thenumber of glycosylation sites, and F equals the fraction of desiredglycoforms. A glycoprotein with three glycosylation sites would have a0.1% chance of providing the appropriate precursors for complex andhybrid N-glycan processing on all of its glycosylation sites, whichlimits the commercial value of such an approach.

Most enzymes that are active in the ER and Golgi apparatus of S.cerevisiae have pH optima that are between 6.5 and 7.5 (see Table 3).All previous approaches to reduce mannosylation by the action ofrecombinant mannosidases have concentrated on enzymes that have a pHoptimum around pH 5.0 (Martinet et al., 1998, and Chiba et al., 1998),even though the activity of these enzymes is reduced to less than 10% atpH 7.0 and thus most likely provide insufficient activity at their pointof use, the ER and early Golgi of P. pastoris and S. cerevisiae. Apreferred process utilizes an o-mannosidase in vivo, where the pHoptimum of the mannosidase is within 1.4 pH units of the average pHoptimum of other representative marker enzymes localized in the sameorganelle(s). The pH optimum of the enzyme to be targeted to a specificorganelle should be matched with the pH optimum of other enzymes foundin the same organelle, such that the maximum activity per unit enzyme isobtained. Table 3 summarizes the activity of mannosidases from varioussources and their respective pH optima. Table 4 summarizes theirlocation. TABLE 3 Mannosidases and their pH optimum. pH Source Enzymeoptimum Reference Aspergillus saitoi 1,2-α-mannosidase 5.0 Ichishima etal., 1999 Biochem. J. 339(Pt 3): 589-597 Trichoderma reesei1,2-α-mannosidase 5.0 Maras et al., 2000 J. Biotechnol. 77(2-3): 255-263Penicillium citrinum 1,2-α-D-mannosidase 5.0 Yoshida et al., 1993Biochem. J. 290(Pt 2): 349-354 Aspergillus nidulans 1,2-α-mannosidase6.0 Eades and Hintz, 2000 Homo sapiens 1,2-α-mannosidase 6.0 IA(Golgi)Homo sapiens IB 1,2-α-mannosidase 6.0 (Golgi) Lepidopteran insect Type I1,2-α-Man₆- 6.0 Ren et al., 1995 Biochem. cells mannosidase 34(8):2489-2495 Homo sapiens α-D-mannosidase 6.0 Chandrasekaran et al., 1984Cancer Res. 44(9): 4059-68 Xanthomonas 1,2,3-α-mannosidase 6.0 manihotisMouse IB (Golgi) 1,2-α-mannosidase 6.5 Schneikert and Herscovics, 1994Glycobiology. 4(4): 445-50 Bacillus sp. (secreted) 1,2-α-D-mannosidase7.0 Maruyama et al., 1994 Carbohydrate Res. 251: 89-98

When one attempts to trim high mannose structures to yield Man₅GlcNAc₂in the ER or the Golgi apparatus of S. cerevisiae, one may choose anyenzyme or combination of enzymes that (1) has/have a sufficiently closepH optimum (i.e. between pH 5.2 and pH 7.8), and (2) is/are known togenerate, alone or in concert, the specific isomeric Man₅GlcNAc₂structure required to accept subsequent addition of GlcNAc by GnT I. Anyenzyme or combination of enzymes that has/have shown to generate astructure that can be converted to GlcNAcMan₅GlcNAc₂ by GnT I in vitrowould constitute an appropriate choice. This knowledge may be obtainedfrom the scientific literature or experimentally by determining that apotential mannosidase can convert Man₈GlcNAc₂-PA to Man₅GlcNAc₂-PA andthen testing, if the obtained Man₅GlcNAc₂-PA structure can serve asubstrate for GnT I and UDP-GlcNAc to give GlcNAcMan₅GlcNAc₂ in vitro.For example, mannosidase IA from a human or murine source would be anappropriate choice.

1,2-Mannosidase Activity in the ER and Golgi

Previous approaches to reduce mannosylation by the action of clonedexogenous mannosidases have failed to yield glycoproteins having asufficient fraction (e.g. >27 mole %) of N-glycans having the structureMan₅GlcNAc₂ (Martinet et al., 1998, and Chiba et al., 1998). Theseenzymes should function efficiently in ER or Golgi apparatus to beeffective in converting nascent glycoproteins. Whereas the twomannosidases utilized in the prior art (from A. saitoi and T. reesei)have pH optima of 5.0, most enzymes that are active in the ER and Golgiapparatus of yeast (e.g. S. cerevisiae) have pH optima that are between6.5 and 7.5 (see Table 3). Since the glycosylation of proteins is ahighly evolved and efficient process, it can be concluded that theinternal pH of the ER and the Golgi is also in the range of about 6-8.At pH 7.0, the activity of the mannosidases used in the prior art isreduced to less than 10%, which is insufficient for the efficientproduction of Man₅GlcNAc₂ in vivo. TABLE 4 Cellular location and pHoptima of various glycosylation-related enzymes of S. cerevisiae. pHGene Activity Location optimum Author(s) Ktr1 α-1,2 Golgi 7.0 Romero etal., mannosyltransferase 1997 Biochem. J. 321(Pt 2): 289-295 Mns1α-1,2-mannosidase ER 6.5 CWH41 glucosidase I ER 6.8 —mannosyltransferase Golgi 7-8 Lehele and Tanner, 1974 Biochim. Biophys.Acta 350(1): 225-235 Kre2 α-1,2 Golgi 6.5-9.0 Romero et al.,mannosyltransferase 1997

The α-1,2-mannosidase enzyme should have optimal activity at a pHbetween 5.1 and 8.0. In a preferred embodiment, the enzyme has anoptimal activity at a pH between 5.9 and 7.5. The optimal pH may bedetermined under in vitro assay conditions. Preferred mannosidasesinclude those listed in Table 3 having appropriate pH optima, e.g.Aspergillus nidulans, Homo sapiens IA(Golgi), Homo sapiens IB (Golgi),Lepidopteran insect cells (IPLB-SF21AE), Homo sapiens, mouse IB (Golgi),and Xanthomonas manihotis. In a preferred embodiment, a single clonedmannosidase gene is expressed in the host organism. However, in somecases it may be desirable to express several different mannosidasegenes, or several copies of one particular gene, in order to achieveadequate production of Man₅GlcNAc₂. In cases where multiple genes areused, the encoded mannosidases should all have pH optima within thepreferred range of 5.1 to 8.0, or especially between 5.9 and 7.5. In anespecially preferred embodiment mannosidase activity is targeted to theER or cis Golgi, where the early reactions of glycosylation occur.

Formation of Complex N-glycans

A second step of the process involves the sequential addition of sugarsto the nascent carbohydrate structure by engineering the expression ofglucosyltransferases into the Golgi apparatus. This process firstrequires the functional expression of GnT I in the early or medial Golgiapparatus as well as ensuring the sufficient supply of UDP-GlcNAc.

Integration Sites

Since the ultimate goal of this genetic engineering effort is a robustprotein production strain that is able to perform well in an industrialfermentation process, the integration of multiple genes into the fungalchromosome involves careful planning. The engineered strain will mostlikely have to be transformed with a range of different genes, and thesegenes will have to be transformed in a stable fashion to ensure that thedesired activity is maintained throughout the fermentation process. Anycombination of the following enzyme activities will have to beengineered into the fungal protein expression host: sialyltransferases,mannosidases, fucosyltransferases, galactosyltransferases,glucosyltransferases, GlcNAc transferases, ER and Golgi specifictransporters (e.g. syn and antiport transporters for UDP-galactose andother precursors), other enzymes involved in the processing ofoligosaccharides, and enzymes involved in the synthesis of activatedoligosaccharide precursors such as UDP-galactose, CMP-N-acetylneuraminicacid. At the same time a number of genes which encode enzymes known tobe characteristic of non-human glycosylation reactions, will have to bedeleted.

Targeting of Glycosyltransferases to Specific Organelles:

Glycosyltransferases and mannosidases line the inner (luminal) surfaceof the ER and Golgi apparatus and thereby provide a “catalytic” surfacethat allows for the sequential processing of glycoproteins as theyproceed through the ER and Golgi network. In fact the multiplecompartments of the cis, medial, and trans Golgi and the trans-GolgiNetwork (TGN), provide the different localities in which the orderedsequence of glycosylation reactions can take place. As a glycoproteinproceeds from synthesis in the ER to full maturation in the late Golgior TGN, it is sequentially exposed to different glycosidases,mannosidases and glycosyltransferases such that a specific carbohydratestructure may be synthesized. Much work has been dedicated to revealingthe exact mechanism by which these enzymes are retained and anchored totheir respective organelle. The evolving picture is complex but evidencesuggests that stem region, membrane spanning region and cytoplasmic tailindividually or in concert direct enzymes to the membrane of individualorganelles and thereby localize the associated catalytic domain to thatlocus.

Targeting sequences are well known and described in the scientificliterature and public databases, as discussed in more detail below withrespect to libraries for selection of targeting sequences and targetedenzymes.

Method for Producing a Library to Produce Modified GlycosylationPathways

A library including at least two genes encoding exogeneous glycosylationenzymes is transformed into the host organism, producing a geneticallymixed population. Transformants having the desired glycosylationphenotypes are then selected from the mixed population. In a preferredembodiment, the host organism is a yeast, especially P. pastoris, andthe host glycosylation pathway is modified by the operative expressionof one or more human or animal glycosylation enzymes, yielding proteinN-glycans similar or identical to human glycoforms. In an especiallypreferred embodiment, the DNA library includes genetic constructsencoding fusions of glycosylation enzymes with targeting sequences forvarious cellular loci involved in glycosylation especially the ER, cisGolgi, medial Golgi, or trans Golgi.

Examples of modifications to glycosylation which can be effected usingmethod are: (1) engineering an eukaryotic microorganism to trim mannoseresidues from Man₈GlcNAc₂ to yield Man₅GlcNAc₂ as a protein N-glycan;(2) engineering an eukaryotic microorganism to add anN-acetylglucosamine (GlcNAc) residue to Man₅GlcNAc₂ by action of GlcNActransferase I; (3) engineering an eukaryotic microorganism tofunctionally express an enzyme such as an N-acetylglucosaminetransferase (GnT I, GnT II, GnT III, GnT IV, GnT V, GnT VI), mannosidaseII, fucosyltransferase, galactosyl tranferase (GalT) orsialyltransferases (ST).

By repeating the method, increasingly complex glycosylation pathways canbe engineered into the target microorganism. In one preferredembodiment, the host organism is transformed two or more times with DNAlibraries including sequences encoding glycosylation activities.Selection of desired phenotypes may be performed after each round oftransformation or alternatively after several transformations haveoccurred. Complex glycosylation pathways can be rapidly engineered inthis manner.

DNA Libraries

It is necessary to assemble a DNA library including at least twoexogenous genes encoding glycosylation enzymes. In addition to the openreading frame sequences, it is generally preferable to provide eachlibrary construct with such promoters, transcription terminators,enhancers, ribosome binding sites, and other functional sequences as maybe necessary to ensure effective transcription and translation of thegenes upon transformation into the host organism. Where the host isPichia pastoris, suitable promoters include, for example, the AOX1,AOX2, DAS, and P40 promoters. It is also preferable to provide eachconstruct with at least one selectable marker, such as a gene to impartdrug resistance or to complement a host metabolic lesion. The presenceof the marker is useful in the subsequent selection of transformants;for example, in yeast the URA3, HIS4, SUC2, G418, BLA, or SHBLE genesmay be used.

In some cases the library may be assembled directly from existing orwild-type genes. In a preferred embodiment however the DNA library isassembled from the fusion of two or more sub-libraries. By the in-frameligation of the sub-libraries, it is possible to create a large numberof novel genetic constructs encoding useful targeted glycosylationactivities. For example, one useful sub-library includes DNA sequencesencoding any combination of enzymes such as sialyltransferases,mannosidases, fucosyltransferases, galactosyltransferases,glucosyltransferases, and GlcNAc transferases. Preferably, the enzymesare of human origin, although other mammalian, animal, or fungal enzymesare also useful. In a preferred embodiment, genes are truncated to givefragments encoding the catalytic domains of the enzymes. By removingendogenous targeting sequences, the enzymes may then be redirected andexpressed in other cellular loci. The choice of such catalytic domainsmay be guided by the knowledge of the particular environment in whichthe catalytic domain is subsequently to be active. For example, if aparticular glycosylation enzyme is to be active in the late Golgi, andall known enzymes of the host organism in the late Golgi have a certainpH optimum, then a catalytic domain is chosen which exhibits adequateactivity at that pH.

Another useful sub-library includes DNA sequences encoding signalpeptides that result in localization of a protein to a particularlocation within the ER, Golgi, or trans Golgi network. These signalsequences may be selected from the host organism as well as from otherrelated or unrelated organisms. Membrane-bound proteins of the ER orGolgi typically may include, for example, N-terminal sequences encodinga cytosolic tail (ct), a transmembrane domain (tmd), and a stem region(sr). The ct, tmd, and sr sequences are sufficient individually or incombination to anchor proteins to the inner (lumenal) membrane of theorganelle. Accordingly, a preferred embodiment of the sub-library ofsignal sequences includes ct, trnd, and/or sr sequences from theseproteins. In some cases it is desirable to provide the sub-library withvarying lengths of sr sequence. This may be accomplished by PCR usingprimers that bind to the 5′ end of the DNA encoding the cytosolic regionand employing a series of opposing primers that bind to various parts ofthe stem region. Still other useful sources of signal sequences includeretrieval signal peptides, e.g. the tetrapeptides HDEL (SEQ ID NO:5) orKDEL (SEQ ID NO:6), which are typically found at the C-terminus ofproteins that are transported retrograde into the ER or Golgi. Stillother sources of signal sequences include (a) type II membrane proteins,(b) the enzymes listed in Table 3, (c) membrane spanning nucleotidesugar transporters that are localized in the Golgi, and (d) sequencesreferenced in Table 5. TABLE 5 Sources of useful compartmental targetingsequences Gene or Location of Gene Sequence Organism Function ProductMnsI S. cerevisiae α-1,2-mannosidase ER OCH1 S. cerevisiae 1,6- Golgi(cis) mannosyltransferase MNN2 S. cerevisiae 1,2- Golgi (medial)mannosyltransferase MNN1 S. cerevisiae 1,3- Golgi (trans)mannosyltransferase OCH1 P. pastoris 1,6- Golgi (cis)mannosyltransferase 2,6 ST H. sapiens 2,6-sialyltransferase trans Golginetwork UDP-Gal T S. pombe UDP-Gal transporter Golgi Mnt1 S. cerevisiae1,2- Golgi (cis) mannosyltransferase HDEL at C- S. cerevisiae retrievalsignal ER terminus (SEQ ID NO: 5)

In any case, it is highly preferred that signal sequences are selectedwhich are appropriate for the enzymatic activity or activities which areto be engineered into the host. For example, in developing a modifiedmicroorganism capable of terminal sialylation of nascent N-glycans, aprocess which occurs in the late Golgi in humans, it is desirable toutilize a sub-library of signal sequences derived from late Golgiproteins. Similarly, the trimming of Man₈GlcNAc₂ by an α-1,2-mannosidaseto give Man₅GlcNAc₂ is an early step in complex N-glycan formation inhumans. It is therefore desirable to have this reaction occur in the ERor early Golgi of an engineered host microorganism. A sub-libraryencoding ER and early Golgi retention signals is used.

In a preferred embodiment, a DNA library is then constructed by thein-frame ligation of a sub-library including DNA encoding signalsequences with a sub-library including DNA encoding glycosylationenzymes or catalytically active fragments thereof. The resulting libraryincludes synthetic genes encoding fusion proteins. In some cases it isdesirable to provide a signal sequence at the N-terminus of a fusionprotein, or in other cases at the C-terminus. In some cases signalsequences may be inserted within the open reading frame of an enzyme,provided the protein structure of individual folded domains is notdisrupted.

The method is most effective when a DNA library transformed into thehost contains a large diversity of sequences, thereby increasing theprobability that at least one transformant will exhibit the desiredphenotype. Accordingly, prior to transformation, a DNA library or aconstituent sub-library may be subjected to one or more rounds of geneshuffling, error prone PCR, or in vitro mutagenesis.

Transformation

The DNA library is then transformed into the host organism. In yeast,any convenient method of DNA transfer may be used, such aselectroporation, the lithium chloride method, or the spheroplast method.To produce a stable strain suitable for high-density fermentation, it isdesirable to integrate the DNA library constructs into the hostchromosome. In a preferred embodiment, integration occurs via homologousrecombination, using techniques known in the art. For example, DNAlibrary elements are provided with flanking sequences homologous tosequences of the host organism. In this manner integration occurs at adefined site in the host genome, without disruption of desirable oressential genes. In an especially preferred embodiment, library DNA isintegrated into the site of an undesired gene in a host chromosome,effecting the disruption or deletion of the gene. For example,integration into the sites of the OCH1, MNN1, or MNN4 genes allows theexpression of the desired library DNA while preventing the expression ofenzymes involved in yeast hypermannosylation of glycoproteins. In otherembodiments, library DNA may be introduced into the host via achromosome, plasmid, retroviral vector, or random integration into thehost genome. In any case, it is generally desirable to include with eachlibrary DNA construct at least one selectable marker gene to allow readyselection of host organisms that have been stably transformed.Recyclable marker genes such as ura3, which can be selected for oragainst, are especially suitable.

Selection Process

After transformation of the host strain with the DNA library,transformants displaying the desired glycosylation phenotype areselected. Selection may be performed in a single step or by a series ofphenotypic enrichment and/or depletion steps using any of a variety ofassays or detection methods. Phenotypic characterization may be carriedout manually or using automated high-throughput screening equipment.Commonly a host microorganism displays protein N-glycans on the cellsurface, where various glycoproteins are localized. Accordingly intactcells may be screened for a desired glycosylation phenotype by exposingthe cells to a lectin or antibody that binds specifically to the desiredN-glycan. A wide variety of oligosaccharide-specific lectins areavailable commercially (EY Laboratories, San Mateo, Calif.).Alternatively, antibodies to specific human or animal N-glycans areavailable commercially or may be produced using standard techniques. Anappropriate lectin or antibody may be conjugated to a reporter molecule,such as a chromophore, fluorophore, radioisotope, or an enzyme having achromogenic substrate (Guillen et al., 1998. Proc. Natl. Acad. Sci. USA95(14): 7888-7892). Screening may then be performed using analyticalmethods such as spectrophotometry, fluorimetry, fluorescence activatedcell sorting, or scintillation counting. In other cases, it may benecessary to analyze isolated glycoproteins or N-glycans fromtransformed cells. Protein isolation may be carried out by techniquesknown in the art. In cases where an isolated N-glycan is required, anenzyme such as endo-β-N-acetylglucosaminidase (Genzyme Co., Boston,Mass.) may be used to cleave the N-glycans from glycoproteins. Isolatedproteins or N-glycans may then be analyzed by liquid chromatography(e.g. HPLC), mass spectroscopy, or other suitable means. U.S. Pat. No.5,595,900 teaches several methods by which cells with desiredextracellular carbohydrate structures may be identified. Prior toselection of a desired transformant, it may be desirable to deplete thetransformed population of cells having undesired phenotypes. Forexample, when the method is used to engineer a functional mannosidaseactivity into cells, the desired transformants will have lower levels ofmannose in cellular glycoprotein. Exposing the transformed population toa lethal radioisotope of mannose in the medium depletes the populationof transformants having the undesired phenotype, i.e. high levels ofincorporated mannose. Alternatively, a cytotoxic lectin or antibody,directed against an undesirable N-glycan, may be used to deplete atransformed population of undesired phenotypes.

Methods for Providing Sugar Nucleotide Precursors to the Golgi Apparatus

For a glycosyltransferase to function satisfactorily in the Golgi, it isnecessary for the enzyme to be provided with a sufficient concentrationof an appropriate nucleotide sugar, which is the high-energy donor ofthe sugar moiety added to a nascent glycoprotein. These nucleotidesugars to the appropriate compartments are provided by expressing anexogenous gene encoding a sugar nucleotide transporter in the hostmicroorganism. The choice of transporter enzyme is influenced by thenature of the exogenous glycosyltransferase being used. For example, aGlcNAc transferase may require a UDP-GlcNAc transporter, afucosyltransferase may require a GDP-fucose transporter, agalactosyltransferase may require a UDP-galactose transporter, or asialyltransferase may require a CMP-sialic acid transporter.

The added transporter protein conveys a nucleotide sugar from thecytosol into the Golgi apparatus, where the nucleotide sugar may bereacted by the glycosyltransferase, e.g. to elongate an N-glycan. Thereaction liberates a nucleoside diphosphate or monophosphate, e.g. UDP,GDP, or CMP. As accumulation of a nucleoside diphosphate inhibits thefurther activity of a glycosyltransferase, it is frequently alsodesirable to provide an expressed copy of a gene encoding a nucleotidediphosphatase. The diphosphatase (specific for UDP or GDP asappropriate) hydrolyzes the diphosphonucleoside to yield a nucleosidemonosphosphate and inorganic phosphate. The nucleoside monophosphatedoes not inhibit the glycotransferase and in any case is exported fromthe Golgi by an endogenous cellular system. Suitable transporterenzymes, which are typically of mammalian origin, are described below.

EXAMPLES

The use of the above general method may be understood by reference tothe following non-limiting examples. Examples of preferred embodimentsare also summarized in Table 6.

Example 1 Engineering of P. pastoris with α-1,2-Mannosidase to ProduceInterferon

An α-1,2-mannosidase is required for the trimming of Man₈GlcNAc₂ toyield Man₅GlcNAc₂, an essential intermediate for complex N-glycanformation. An OCH1 mutant of P. pastoris is engineered to expresssecreted human interferon-β under the control of an aox promoter. A DNAlibrary is constructed by the in-frame ligation of the catalytic domainof human mannosidase IB (an α-1,2-mannosidase) with a sub-libraryincluding sequences encoding early Golgi localization peptides. The DNAlibrary is then transformed into the host organism, resulting in agenetically mixed population wherein individual transformants eachexpress interferon-β as well as a synthetic mannosidase gene from thelibrary. Individual transformant colonies are cultured and theproduction of interferon is induced by addition of methanol. Under theseconditions, over 90% of the secreted protein includes interferon-β.Supernatants are purified to remove salts and low-molecular weightcontaminants by C₁₈ silica reversed-phase chromatography. Desiredtransformants expressing appropriately targeted, activeα-1,2-mannosidase produce interferon-, including N-glycans of thestructure Man₅GlcNAc₂, which has a reduced molecular mass compared tothe interferon of the parent strain. The purified supernatants includinginterferon-β are analyzed by MALDI-TOF mass spectroscopy and coloniesexpressing the desired form of interferon-β are identified.

Example 2 Engineering of Strain to Express GlcNAc Transferase I

GlcNAc Transferase I activity is required for the maturation of complexN-glycans. Man₅GlcNAc₂ may only be trimmed by mannosidase II, anecessary step in the formation of human glycoforms, after the additionof GlcNAc to the terminal α-1,3mannose residue by GlcNAc Transferase I(Schachter, 1991 Glycobiology 1(5):453-461). Accordingly a library isprepared including DNA fragments encoding suitably targeted GlcNAcTransferase I genes. The host organism is a strain, e.g. a yeast, thatis deficient in hypermannosylation (e.g. an OCH1 mutant), provides thesubstrate UDP-GlcNAc in the Golgi and/or ER, and provides N-glycans ofthe structure Man₅GlcNAc₂ in the Golgi and/or ER. After transformationof the host with the DNA library, the transformants are screened forthose having the highest concentration of terminal GlcNAc on the cellsurface, or alternatively secrete the protein having the highestterminal GlcNAc content. Such a screen is performed using a visualmethod (e.g. a staining procedure), a specific terminal GlcNAc bindingantibody, or a lectin. Alternatively the desired transformants exhibitreduced binding of certain lectins specific for terminal mannoseresidues.

Example 3 Engineering of Strains with a Mannosidase II

In another example, it is desirable in order to generate a humanglycoform in a microorganism to remove the two remaining terminalmannoses from the structure GlcNAcMan₅GlcNAc₂ by action of a mannosidaseII. A DNA library including sequences encoding cis and medial Golgilocalization signals is fused in-frame to a library encoding mannosidaseII catalytic domains. The host organism is a strain, e.g. a yeast, thatis deficient in hypermannosylation (e.g. an OCH1 mutant) and providesN-glycans having the structure GlcNAcMan₅GlcNAc₂ in the Golgi and/or ER.After transformation, organisms having the desired glycosylationphenotype are selected. An in vitro assay is used in one method. Thedesired structure GlcNAcMan₃GlcNAc₂ (but not the undesiredGlcNAcMan₅GlcNAc₂) is a substrate for the enzyme GlcNAc Transferase II.Accordingly, single colonies may be assayed using this enzyme in vitroin the presence of the substrate, UDP-GlcNAc. The release of UDP isdetermined either by HPLC or an enzymatic assay for UDP. Alternativelyradioactively labeled UDP-GlcNAc is used.

The foregoing in vitro assays are conveniently performed on individualcolonies using high-throughput screening equipment. Alternatively alectin binding assay is used. In this case the reduced binding oflectins specific for terminal mannoses allows the selection oftransformants having the desired phenotype. For example, Galantusnivalis lectin binds specifically to terminal α-1,3-mannose, theconcentration of which is reduced in the presence of operativelyexpressed mannosidase II activity. In one suitable method, G. nivalislectin attached to a solid agarose support (available from SigmaChemical, St. Louis, Mo.) is used to deplete the transformed populationof cells having high levels of terminal α-1,3-mannose.

Example 4 Engineering of Organisms to Express Sialyltransferase

The enzymes α2,3-sialyltransferase and α2,6-sialyltransferase addterminal sialic acid to galactose residues in nascent human N-glycans,leading to mature glycoproteins. In human the reactions occur in thetrans Golgi or TGN. Accordingly a DNA library is constructed by thein-frame fusion of sequences encoding sialyltransferase catalyticdomains with sequences encoding trans Golgi or TGN localization signals.The host organism is a strain, e.g. a yeast, that is deficient inhypermannosylation (e.g. an OCH1 mutant), which provides N-glycanshaving terminal galactose residues in the trans Golgi or TGN, andprovides a sufficient concentration of CMP-sialic acid in the transGolgi or TGN. Following transformation, transformants having the desiredphenotype are selected using a fluorescent antibody specific forN-glycans having a terminal sialic acid.

Example 5 Method of Engineering Strains to Express UDP-GlcNAcTransporter

The cDNA of human Golgi UDP-GlcNAc transporter has been cloned by Ishidaand coworkers. (Ishida, N., et al. 1999 J. Biochem. 126(1): 68-77.Guillen and coworkers have cloned the canine kidney Golgi UDP-GlcNActransporter by phenotypic correction of a Kluyveromyces lactis mutantdeficient in Golgi UDP-GlcNAc transport. (Guillen, E., et al. 1998).Thus a mammalian Golgi UDP-GlcNAc transporter gene has all of thenecessary information for the protein to be expressed and targetedfunctionally to the Golgi apparatus of yeast.

Example 6 Method of Engineering Strains to Express GDP-FucoseTransporter

The rat liver Golgi membrane GDP-fucose transporter has been identifiedand purified by Puglielli, L. and C. B. Hirschberg 1999 J. Biol. Chem.274(50):35596-35600. The corresponding gene can be identified usingstandard techniques, such as N-terminal sequencing and Southern blottingusing a degenerate DNA probe. The intact gene can is then be expressedin a host microorganism that also expresses a fucosyltransferase.

Example 7 Method of Engineering Strains to Express UDP-GalactoseTransporter

Human UDP-galactose (UDP-Gal) transporter has been cloned and shown tobe active in S. cerevisiae. (Kainuma, M., et al. 1999 Glycobiology 9(2):133-141). A second human UDP-galactose transporter (hUGT1) has beencloned and functionally expressed in Chinese Hamster Ovary Cells. Aoki,K., et al. 1999 J. Biochem. 126(5): 940-950. Likewise Segawa andcoworkers have cloned a UDP-galactose transporter fromSchizosaccharomyces pombe (Segawa, H., et al. 1999 Febs Letters 451(3):295-298).

CMP-Sialic Acid Transporter

Human CMP-sialic acid transporter (hCST) has been cloned and expressedin Lec 8 CHO cells by Aoki and coworkers (1999). Molecular cloning ofthe hamster CMP-sialic acid transporter has also been achieved (Eckhardtand Gerardy Schahn 1997 Eur. J. Biochem. 248(1): 187-192). Thefunctional expression of the murine CMP-sialic acid transporter wasachieved in Saccharomyces cerevisiae by Berninsone, P., et al. 1997 J.Biol. Chem. 272(19):12616-12619. TABLE 6 Examples of preferredembodiments of the methods for modifying glycosylation in a eukaroyticmicroorganism, e.g. Pichia pastoris Suitable Suitable SuitableTransporters Desired Catalytic Suitable Sources of Gene and/or StructureActivities Localization Sequences Deletions Phosphatases Man₅GlcNAc₂α-1,2- Mns1 (N-terminus, OCH1 none mannosidase S. cerevisiae) MNN4(murine, Och1 (N-terminus, MNN6 human, S. cerevisiae, P. pastoris)Bacillus sp., Ktr1 A. nidulans) Mnn9 Mnt1 (S. cerevisiae) KDEL (SEQ IDNO: 6), HDEL (SEQ ID NO: 5) (C-terminus) GlcNAcMan₅GlcNAc₂ GlcNAc Och1(N-terminus, OCH1 UDP-GlcNAc Transferase S. cerevisiae, P. pastoris)MNN4 transporter I, (human, KTR1 (N-terminus) MNN6 (human, murine,murine, rat KDEL (SEQ ID NO: 6), K. lactis) etc.) HDEL (SEQ ID NO: 5)UDPase (human) (C-terminus) Mnn1 (N-terminus, S. cerevisiae) Mnt1(N-terminus, S. cerevisiae) GDPase (N-terminus, S. cerevisiae)GlcNAcMan₃GlcNAc₂ mannosidase Ktr1 OCH1 UDP-GlcNAc II Mnn1 (N-terminus,MNN4 transporter S. cerevisiae) MNN6 (human, murine, Mnt1(N-terminus, K.lactis) S. cerevisiae) UDPase (human) Kre2/Mnt1 (S. cerevisiae) Kre2 (P.pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae)GlcNAc₍₂₋₄₎ GlcNAc Mnn1 (N-terminus, OCH1 UDP-GlcNAc Man₃GlcNAc₂Transferase S. cerevisiae) MNN4 transporter II, III, IV, V Mnt1(N-terminus, MNN6 (human, murine, (human, S. cerevisiae) K. lactis)murine) Kre2/Mnt1 (S. cerevisiae) UDPase (human) Kre2 (P. pastoris) Ktr1(S. cerevisiae) Ktr1 (P. pastoris) Mnn1 (S. cerevisiae)Gal₍₁₋₄₎GlcNAc₍₂₋₄₎- β-1,4- Mnn1 (N-terminus, OCH1 UDP-GalactoseMan₃GlcNAc₂ Galactosyl S. cerevisiae) MNN4 transporter transferaseMnt1(N-terminus, MNN6 (human, (human) S. cerevisiae) S. pombe) Kre2/Mnt1(S. cerevisiae) Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P.pastoris) Mnn1 (S. cerevisiae) NANA₍₁₋₄₎- α-2,6- KTR1 OCH1 CMP-Sialicacid Gal₍₁₋₄₎GlcNAc₍₂₋₄₎- Sialyltransferase MNN1 (N-terminus, MNN4transporter Man₃GlcNAc₂ (human) S. cerevisiae) MNN6 (human) α-2,3- MNT1(N-terminus, Sialyltransferase S. cerevisiae) Kre2/Mnt1 (S. cerevisiae)Kre2 (P. pastoris) Ktr1 (S. cerevisiae) Ktr1 (P. pastoris) MNN1 (S.cerevisiae)

TABLE 7 DNA and Protein Sequence Resources  1. European BioinformaticsInstitute (EBI) is a centre for research and services in   bioinformatics  2. Swissprot database  3. List of knownglycosyltransferases and their origin. β1,2 (GnT I) EC 2.4.1.101  4.human cDNA, Kumar et al (1990) Proc. Natl. Acad. Sci. USA 87: 9948-9952 5. human gene, Hull et al (1991) Biochem. Biophys. Res. Commun. 176:608-615  6. mouse cDNA, Kumar et al (1992) Glycobiology 2: 383-393  7.mouse gene, Pownall et al (1992) Genomics 12: 699-704  8. murine gene(5′ flanking, non-coding), Yang et al (1994) Glycobiology 5: 703-712  9.rabbit cDNA, Sarkar et al (1991) Proc. Natl. Acad. Sci. USA 88: 234-23810. rat cDNA, Fukada et al (1994) Biosci.Biotechnol.Biochem. 58: 200-2011,2 (GnT II) EC 2.4.1.143 11. human gene, Tan et al (1995) Eur. J.Biochem. 231: 317-328 12. rat cDNA, D′Agostaro et al (1995) J. Biol.Chem. 270: 15211-15221 13. β1,4 (GnT III) EC 2.4.1.144 14. human cDNA,Ihara et al (1993) J. Biochem.113: 692-698 15. murine gene, Bhaumik etal (1995) Gene 164: 295-300 16. rat cDNA, Nishikawa et al (1992) J.Biol. Chem. 267: 18199-18204 β1,4 (GnT IV) EC 2.4.1.145 17. human cDNA,Yoshida et al (1998) Glycoconjugate Journal 15: 1115-1123 18. bovinecDNA, Minowa et al., European Patent EP 0 905 232    β1,6 (GnT V) EC2.4.1.155 19. human cDNA, Saito et al (1994) Biochem. Biophys. Res.Commun. 198: 318-327 20. rat cDNA, Shoreibah et al (1993) J. Biol. Chem.268: 15381-15385 β1,4 Galactosyltransferase, EC 2.4.1.90 (LacNAcsynthetase) EC 2.4.1.22 (lactose synthetase) 21. bovine cDNA, D′Agostaroet al (1989) Eur. J. Biochem. 183: 211-217 22. bovine cDNA (partial),Narimatsu et al (1986) Proc. Natl. Acad. Sci. USA    83: 4720-4724 23.bovine cDNA (partial), Masibay & Qasba (1989) Proc. Natl. Acad. Sci. USA   86: 5733-5377 24. bovine cDNA (5′ end), Russo et al (1990) J. Biol.Chem. 265: 3324 25. chicken cDNA (partial), Ghosh et al (1992) Biochem.Biophys. Res. Commun.    1215-1222 26. human cDNA, Masri et al (1988)Biochem. Biophys. Res. Commun. 157: 657-663 27. human cDNA, (HeLa cells)Watzele & Berger (1990) Nucl. Acids Res. 18: 7174 28. human cDNA,(partial) Uejima et al (1992) Cancer Res. 52: 6158-6163 29. human cDNA,(carcinoma) Appert et al (1986) Biochem. Biophys. Res. Commun.    139:163-168 30. human gene, Mengle-Gaw et al (1991) Biochem. Biophys. Res.Commun.    176: 1269-1276 31. murine cDNA, Nakazawa et al (1988) J.Biochem. 104: 165-168 32. murine cDNA, Shaper et al (1988) J. Biol.Chem. 263: 10420-10428 33. murine cDNA (novel), Uehara & Muramatsuunpublished 34. murine gene, Hollis et al (1989) Biochem. Biophys. Res.Commun. 162: 1069-1075 35. rat protein (partial), Bendiak et al (1993)Eur. J. Biochem. 216: 405-417 2,3-Sialyltransferase, (ST3Gal II)(N-linked) (Gal-1,3/4-GlcNAc) EC 2.4.99.6 36. human cDNA, Kitagawa &Paulson (1993) Biochem. Biophys. Res. Commun.    194: 375-382 37. ratcDNA, Wen et al (1992) J. Biol. Chem. 267: 21011-210192,6-Sialyltransferase, (ST6Gal I) EC 2.4.99.1 38. chicken, Kurosawa etal (1994) Eur. J. Biochem 219: 375-381 39. human cDNA (partial), Lanceet al (1989) Biochem. Biophys. Res. Commun.    164: 225-232 40. humancDNA, Grundmann et al (1990) Nucl. Acids Res. 18: 667 41. human cDNA,Zettlmeisl et al (1992) Patent EPO475354-A/3 42. human cDNA, Stamenkovicet al (1990) J. Exp. Med. 172: 641-643 (CD75) 43. human cDNA, Bast et al(1992) J. Cell Biol. 116: 423-435 44. human gene (partial), Wang et al(1993) J. Biol. Chem. 268: 4355-4361 45. human gene (5′ flank), Aasheimet al (1993) Eur. J. Biochem. 213: 467-475 46. human gene (promoter),Aas-Eng et al (1995) Biochim. Biophys. Acta 1261: 166-169 47. mousecDNA, Hamamoto et al (1993) Bioorg. Med. Chem. 1: 141-145 48. rat cDNA,Weinstein et al (1987) J. Biol. Chem. 262: 17735-17743 49. rat cDNA(transcript fragments), Wang et al (1991) Glycobiology 1: 25-31, Wang   et al (1990) J. Biol. Chem. 265: 17849-17853 50. rat cDNA (5′ end),O′Hanlon et al (1989) J. Biol. Chem. 264: 17389-17394; Wang    et al(1991) Glycobiology 1: 25-31 51. rat gene (promoter), Svensson et al(1990) J. Biol. Chem. 265: 20863-20688 52. rat mRNA (fragments), Wen etal (1992) J. Biol. Chem. 267: 2512-2518

Additional methods and reagents which can be used in the methods formodifying the glycosylation are described in the literature, such asU.S. Pat. No. 5,955,422, U.S. Pat. No. 4,775,622, U.S. Pat. No.6,017,743, U.S. Pat. No. 4,925,796, U.S. Pat. No. 5,766,910, U.S. Pat.No. 5,834,251, U.S. Pat. No. 5,910,570, U.S. Pat. No. 5,849,904, U.S.Pat. No. 5,955,347, U.S. Pat. No. 5,962,294, U.S. Pat. No. 5,135,854,U.S. Pat. No. 4,935,349, U.S. Pat. No. 5,707,828, and U.S. Pat. No.5,047,335.

Appropriate yeast expression systems can be obtained from sources suchas the American Type Culture Collection, Rockville, Md. Vectors arecommercially available from a variety of sources.

1.-51. (canceled)
 52. A composition comprising a recombinantglycoprotein, wherein at least 30% of the glycoprotein in thecomposition comprises a core oligosaccharide structure consisting ofMan5GlcNAc2 suitable to accept in vivo at least one terminal residue bythe action of a glycosyltransferase to form a hybrid N-glycan.
 53. Thecomposition of claim 52, wherein at least 50% to 100% of theglycoprotein in the composition comprises a core oligosaccharidestructure consisting of Man5GlcNAc2 suitable to accept in vivo at leastone terminal residue by the action of a glycosyltransferase to form ahybrid N-glycan.
 54. A composition comprising a recombinantglycoprotein, wherein the composition comprises a high yield ofglycoprotein having a core oligosaccharide structure consisting ofMan5GlcNAc2 suitable to accept in vivo at least one terminal residue bythe action of a glycosyltransferase to form a hybrid N-glycan.
 55. Thecomposition of claim 52 or 54, wherein the terminal residue is GlcNAc.56. The composition of claim 52 or 54, wherein the glycosyltransferaseis GlcNAc transferase.
 57. The composition of claim 52 or 54, whereinthe hybrid N-glycan is GlcNAcMan5GlcNAc2.
 58. The composition of claim52 or 54, wherein the action further comprises the action of a GlcNActransporter.
 59. A composition comprising a recombinant glycoprotein,wherein the composition comprises a high yield of glycoprotein having acore oligosaccharide structure consisting of GlcNAcMan5GlcNAc2, whereinthe glycoprotein is a suitable substrate to be trimmed in vivo by theaction of a mannosidase to form a complex N-glycan.
 60. The compositionof claim 59, wherein the complex N-glycan is GlcNAcMan3GlcNAc2.
 61. Acomposition comprising a recombinant glycoprotein, wherein thecomposition comprises a high yield of glycoprotein having a coreoligosaccharide structure of GlcNAcMan3GlcNAc2.
 62. A compositioncomprising a recombinant glycoprotein, wherein the composition comprisesa high yield of glycoprotein having a core oligosaccharide structureconsisting of GlcNAc(2-4)Man3GlcNAc2.
 63. The composition of claim 62,wherein the core oligosaccharide structure is produced in vivo by theaction of a glycosyltransferase that is GlcNAc transferase I, II, III,IV, V, or a combination thereof.
 64. The composition of claim 63,wherein the action is in the presence UDP-GlcNAc transporter.
 65. Acomposition comprising a recombinant glycoprotein, wherein thecomposition comprises a high yield of glycoprotein having a coreoligosaccharide structure consisting of Gal(1 -4)GlcNAc(2-4)Man3GlcNAc2.66. The composition of claim 65, wherein the core oligosaccharidestructure is produced in vivo by the action of a β1,4-galactosyltransferase.
 67. The composition of claim 66, wherein the action is inthe presence of a UDP-galactose transporter.
 68. A compositioncomprising a recombinant glycoprotein, wherein the composition comprisesa high yield of glycoprotein having a core oligosaccharide structureconsisting of NANA(1-4)Gal(1-4)GlcNAc(2-4) Man3GlcNAc2.
 69. Thecomposition of claim 68, wherein the core oligosaccharide structure isproduced in vivo by the action of a transferase that isα2,6sialyltransferase, α2,3sialyltransferase, or a combination thereof.70. The composition of claim 69, wherein the action is in the presenceof a CMP-sialic acid transporter.
 71. A composition comprising arecombinant glycoprotein, wherein the composition comprises a high yieldof glycoprotein having a core oligosaccharide structure wherein the coreconsists of GlcNac2 and has fewer than 4 mannose residues and optionallyone or more terminal sugar residues.
 72. A composition comprising arecombinant glycoprotein, wherein the composition comprises a high yieldof glycoprotein having a core oligosaccharide structure wherein the coreconsists of Man3GlcNAc2 and optionally one or more terminal sugarresidues.
 73. The composition of claim 71 or 72, wherein the one or moreterminal sugar residues is selected from the group consisting of GlcNac,Gal, and Sialic acid.
 74. The glycoprotein of any one of the precedingclaims, wherein the glycoprotein is obtained from a cell comprisingreduced high mannose activity.
 75. The glycoprotein of any one of thepreceding claims, wherein the glycoprotein is in the presence of reducedhigh mannose activity achieved by a gene disruption of one or moreenzymes selected from the group consisting of a mannosyltransferase anda mannose transporter.
 76. The glycoprotein of any one of the precedingclaims, wherein the glycoprotein is obtained from a lower eukaryote. 77.The glycoprotein of claim 76, wherein the lower eukaryote is yeast. 78.The glycoprotein of claim 77, wherein the yeast is Pichia.
 79. Theglycoprotein of claim 76, wherein the lower eukaryote is a filamentousfungus.
 80. The recombinant glycoprotein of any of the above claims,wherein the core oligosaccharide structure is further modified by afucosyltransferase to comprise at least one core fucose residue.
 81. Arecombinant glycoprotein obtained from a yeast having a coreoligosaccharide structure consisting of a structure selected from thegroup consisting of Man3GlcNAc2, GlcNAcMan3GlcNAc2,GlcNAc(2-4)Man3GlcNAc2, Gal(1-4)GlcNAc(2-4)Man3GlcNAc2, and NANA(1-4)Gal(1 -4)GlcNAc(2-4) Man3 GlcNAc2.
 82. The recombinant glycoproteinof claim 81, wherein the core oligosaccharide structure is Man3GlcNAc2.83. The recombinant glycoprotein of claim 82, wherein the coreoligosaccharide structure is further modified to comprise at least oneterminal sugar residue.
 84. A recombinant glycoprotein obtained from ayeast having a core oligosaccharide structure wherein the core consistsof Man3GlcNAc2 and optionally one or more terminal sugar residues. 85.The recombinant glycoprotein of any of the above claims, wherein therecombinant glycoprotein is a therapeutic glycoprotein.
 86. Therecombinant glycoprotein of any of the above claims, wherein therecombinant glycoprotein is a human glycoprotein.
 87. The recombinantglycoprotein of any one of the above claims, wherein the glycoprotein isan antibody.
 88. The recombinant glycoprotein of claim 87, wherein theantibody is an IgG or IgM antibody.